A central region of Ku80 mediates interaction with Ku70 in vivo.
Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively . Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo . A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.
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Life Sciences Division, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87545, USA.
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D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004265:DNA Helicases; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D000939:Epitopes / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000939:Epitopes; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C081647:high affinity DNA-binding factor, S cerevisiae; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen